Identification of tyrosine-phosphorylated proteins associated with the nuclear envelope

Abstract

The nuclear envelope separates the nucleoplasm from the rest of the cell. Throughout the cell cycle, its structural integrity is controlled by reversible protein phosphorylation. Whereas its phosphorylation-dependent disassembly during mitosis is well characterized, little is known about phosphorylation events at this structure during interphase. The few characterized examples cover protein phosphorylation at serine and threonine residues, but not tyrosine phosphorylation at the nuclear envelope.Here, we demonstrate that tyrosine phosphorylation and dephosphorylation occur at the nuclear envelope of intact Neuro2a mouse neuroblastoma cells. Tyrosine kinase and phosphatase activities remain associated with purified nuclear envelopes. A similar pattern of tyrosinephosphorylated nuclear envelope proteins suggests that the same tyrosine kinases act at the nuclear envelope of intact cells and at the purified nuclear envelope.We have also identified eight tyrosine-phosphorylated nuclear envelope proteins by 2D BAC/SDS/PAGE, immunoblotting with phosphotyrosine-specific antibodies, tryptic in-gel digestion, and MS analysis of tryptic peptides. These proteins are the lamina proteins lamin A, lamin B1, and lamin B2, the inner nuclear membrane protein LAP2b, the heat shock protein hsc70, and the DNA /RNA-binding proteins PSF, hypothetical 16-kDa protein, and NonO, which copurify with the nuclear envelope.Keywords: lamin; lamina-associated polypeptide 2 (LAP2); NonO; nuclear envelope; tyrosine phosphorylation.The nuclear envelope consists of an outer nuclear membrane, which is continuous with the membrane of the endoplasmic reticulum, an inner nuclear membrane, which is attached to and supported by the nuclear lamina, a network of intermediate filament proteins called lamins, and the pore membrane, which connects the inner and outer membrane at the nuclear pore complexes [1]. The outer nuclear membrane probably resembles the endoplasmic reticulum membrane with regard to its protein composition [2]. The inner membrane harbors proteins that are exclusively located to this membrane. So far, very few proteins have been specifically assigned to this membrane. Examples are the lamin B receptor [3], several isoforms of lamina-associated polypeptides (LAPs) [4±6], Emerin [7], Nurim [8], and MAN-1 [9]. Most of these proteins bind to either lamins or chromatin, or both, thereby shaping the border between nucleoplasm and nuclear envelope and taking part in structuring the chromatin [10]. Balanced proportions of these proteins are necessary for the normal shape and function of the cell nucleus. Overexpression of the lamin B receptor, for example, leads to a deformed nucleus [11]. The nuclear envelope folds irregularly into the nucleus, which appears to be accompanied by a disturbed functional organization of the nucleus.The structure of the nuclear envelope is controlled and stabilized by phosphorylation and dephosphorylation of its proteins. Best characterized are phosphorylation events during mitosis. At the begin of mitosis, the nuc...

Proteomic Analysis of Protein Phosphorylations in Heat Shock Response and Thermotolerance

“…major substrates for retrovirus encoded PTK pp60 v-Src (31), platelet-derived growth factor (32), insulin (33), and hepatocyte growth factor/scatter factor (34) receptor tyrosine kinases. Hsp70 was revealed as phosphorylation at Tyr-524 in COS-1 cells that corresponded with Tyr-525 in mouse Hsp70 (35). Aldolase 1A and lactate dehydrogenase A were shown that phosphorylated in Tyr-361 (Tyr-364 in mouse aldolase 1A) (36) and Tyr-238 (Tyr-239 in mouse lactate dehydrogenase A) (37), respectively.…”

“…However, as the programs use unique algorithms for prediction of phosphorylation sites, combining and comparing the results from the three programs should give useful information. In fact, the programs can predict the tyrosine phosphorylation sites as reported experimentally; HSC70 (spots 1 and 2), aldolase A isoform (spot 65), and lactate dehydrogenase A (spot 76) were previously known as tyrosine-phosphorylated proteins and Tyr-525 (Tyr-524 in COS-1 cells) (35), Tyr-364 (Tyr-361 in rabbit liver cell) (36), and Tyr-239 (Tyr-238 in Rous sarcoma virus-transformed cell) (37) were the tyrosine phosphorylation sites, respectively. At least two of the three programs predicted the known sites correctly.…”

Transcriptional Repressor Germ Cell-less (GCL) and Barrier to Autointegration Factor (BAF) Compete for Binding to Emerin in Vitro

“…Our biochemical analysis of recombinant proteins suggests that emerin can form stable complexes with lamin A and GCL, but only in the absence of BAF. Because many nuclear envelope proteins are differentially phosphorylated during interphase, including lamin A, LAP2␤, and emerin (29,30), we propose that GCL binding to emerin or displacement of BAF from BAF-emerinlamin complexes is regulated by signal transduction in vivo. This predicted interplay between GCL and BAF for binding to emerin will be interesting to explore in living cells, where binding affinities may be further influenced by chromatin.…”

Identification of tyrosine‐phosphorylation sites in the nuclear membrane protein emerin

“…Nuclei and nuclear envelopes were prepared from N2a cells [42]. Ten minutes before the cells were harvested for nuclear preparation, the selective tyrosine‐phosphatase inhibitor potassium‐(2,2′‐bipyridine)‐oxobisperoxovanadate (BiPy) [31] was added to the culture medium at a concentration of 100 µ m .…”